Primary immunodeficiency diseases (PIDDs) are a large group of genetic disorders of the immune system. These disorders vary in the severity and spectrum of symptoms, but without effective and early treatment, they can be fatal. The current approach to SCID utilizes quantitative PCR to detect T cell Receptor Excision Circles (TREC) which is applicable only to a subset of immunodeficiencies. The goal of our proposal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple PIDDs using dried blood spots (DBS). We previously developed a novel proteomic screening method using Selected Reaction Monitoring-Mass Spectrometry (SRM-MS) to simultaneously identify specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3?) and the intracellular proteins, Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and X-linked Agammaglobulinemia (XLA). The objective of this application is to improve the sensitivity of our novel approach by developing peptide immunoaffinity enrichment coupled to SRM-MS (immuno-SRM-MS) to quantify a panel of biomarkers in DBS to facilitate the early detection and diagnosis of multiple life-threatening PIDDs. Our Aims are to: 1. Expand the existing panel of screenable PIDDs by identifying proteotypic signature peptides for 8 additional conditions using SRM-MS. These PIDDs include ADA-deficient SCID, MHC class II deficient SCID, DOCK8 deficiency, Common Variable Immunodeficiency, Ataxia Telangiectasia, Familial hemophagocytic lymphohistiocytosis 2, X-linked lymphoproliferative syndrome, and X-linked chronic granulomatous disease. We will use human cell lines to select signature peptides for these PIDDs and fully optimize SRM-MS conditions. 2. Increase sensitivity of the SRM-MS assay for PIDDs by coupling it with peptide immunoaffinity enrichment. We will employ immuno-SRM procedures for measurements of signature peptides for the target proteins in DBS for various PIDDs to improve the sensitivity of our assay. We will measure performance metrics for each assay by generating a response curve. 3. Evaluate the ability of a multiplex immuno-SRM approach to correctly identify patients with specific immunodeficiencies in a large set of clinical samples. Our multiplexed immuno-SRM assay will be tested on patient DBS samples collected by the Seattle Children's Immunology Diagnostic Laboratory. Positive newborn DBS from WA State will be retrieved and tested.